Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence

Abstract G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra...

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Autores principales:	Dennis D. Fernandes, Chris Neale, Gregory-Neal W. Gomes, Yuchong Li, Aimen Malik, Aditya Pandey, Alexander P. Orazietti, Xudong Wang, Libin Ye, R. Scott Prosser, Claudiu C. Gradinaru
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1d5a6aaa4ef3450b9bd0c50f9ac512ec
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spelling	oai:doaj.org-article:1d5a6aaa4ef3450b9bd0c50f9ac512ec2021-12-02T17:05:12ZLigand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence10.1038/s41598-021-84069-02045-2322https://doaj.org/article/1d5a6aaa4ef3450b9bd0c50f9ac512ec2021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84069-0https://doaj.org/toc/2045-2322Abstract G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.Dennis D. FernandesChris NealeGregory-Neal W. GomesYuchong LiAimen MalikAditya PandeyAlexander P. OraziettiXudong WangLibin YeR. Scott ProsserClaudiu C. GradinaruNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-16 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dennis D. Fernandes Chris Neale Gregory-Neal W. Gomes Yuchong Li Aimen Malik Aditya Pandey Alexander P. Orazietti Xudong Wang Libin Ye R. Scott Prosser Claudiu C. Gradinaru Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence
description	Abstract G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.
format	article
author	Dennis D. Fernandes Chris Neale Gregory-Neal W. Gomes Yuchong Li Aimen Malik Aditya Pandey Alexander P. Orazietti Xudong Wang Libin Ye R. Scott Prosser Claudiu C. Gradinaru
author_facet	Dennis D. Fernandes Chris Neale Gregory-Neal W. Gomes Yuchong Li Aimen Malik Aditya Pandey Alexander P. Orazietti Xudong Wang Libin Ye R. Scott Prosser Claudiu C. Gradinaru
author_sort	Dennis D. Fernandes
title	Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence
title_short	Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence
title_full	Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence
title_fullStr	Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence
title_full_unstemmed	Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence
title_sort	ligand modulation of the conformational dynamics of the a2a adenosine receptor revealed by single-molecule fluorescence
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/1d5a6aaa4ef3450b9bd0c50f9ac512ec
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Ligand modulation of the conformational dynamics of the A2A adenosine receptor revealed by single-molecule fluorescence

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